Combination treatment in vitro with Nutlin, a small-molecule antagonist of MDM2, and pegylated interferon-α 2a specifically targets JAK2V617F-positive polycythemia vera cells

Abstract

Interferon (IFN-α) is effective therapy for polycythemia vera (PV) patients, but it is frequently interrupted because of adverse events. To permit the long-term use of IFN, we propose combining low doses of IFN with Nutlin-3, an antagonist of MDM2, which is also capable of promoting PV CD34(+) cell apoptosis. Combination treatment with subtherapeutic doses of Peg IFN-α 2a and Nutlin-3 inhibited PV CD34(+) cell proliferation by 50% while inhibiting normal CD34(+) cells by 30%. Combination treatment with Nutlin-3 and Peg IFN-α 2a inhibited PV colony formation by 55%-90% while inhibiting normal colony formation by 22%-30%. The combination of these agents also decreased the proportion of JAK2V617F-positive hematopoietic progenitor cells in 6 PV patients studied. Treatment with low doses of Peg IFN-α 2a combined with Nutlin-3 increased phospho-p53 and p21 protein levels in PV CD34(+) cells and increased the degree of apoptosis. These 2 reagents affect the tumor suppressor p53 through different pathways with Peg IFN-α 2a activating p38 MAP kinase and STAT1, leading to increased p53 transcription, whereas Nutlin-3 prevents the degradation of p53. These data suggest that treatment with low doses of both Nutlin-3 combined with Peg IFN-α 2a can target PV hematopoietic progenitor cells, eliminating the numbers of malignant hematopoietic progenitor cells.

Figure 1

PV CD34⁺ cells contained higher levels of MDM2 protein. (A) Western blotting demonstrated the increased expression of MDM2 and lower levels of p53 in PV CD34⁺ cells (7 PVs and 5 normal BMs). (B) The quantification of protein levels was performed densitometrically and normalized to GAPDH levels.

Figure 2

PV CD34⁺ cells responded to the treatment of Nutlin-3. Effects of increasing concentrations of Nutlin-3 on CFU-GM– and BFU-E–derived colony formation by normal bone marrow (A) and PV (B) CD34⁺ cells.

Figure 3

Low doses of Peg IFN-α 2a combined with Nutlin-3 significantly inhibited the proliferation of PV CD34⁺ cells. (A) After treatment with a low dose of Peg IFN-α 2a combined with Nutlin-3, the percentage of reduction in CD34⁺ cell numbers from PV and normal BM compared with cytokines alone. *P < .05. **P < .01. (B) Effects of 200 U/mL of Peg IFN-α 2a combined with 200nM of Nutlin-3 on CFU-GM- and BFU-E-derived colony formation by normal BM CD34⁺ cells. *P < .05. n = 7. (C) Effects of 200 U/mL of Peg IFN-α 2a combined with 200nM of Nutlin-3 on CFU-GM- and BFU-E-derived colony formation by PV CD34⁺ cells. *P < .05. **P < .01. n = 17.

Figure 4

Low doses of Nutlin-3 enhance the effects of a low dose of Peg IFN-α 2a on the apoptosis of PV hematopoietic progenitor cells. Flow cytometric analysis of normal and PV CD34⁺ cells after 4 days of treatment with 200nM of Nutlin-3 alone or 200 U/mL of Peg IFN-α 2a alone or in combination. None of the treatments increased the number of apoptotic cells in cultures of normal CD34⁺ cells (n = 4). By contrast, Peg IFN-α 2a and Nutlin-3 alone increased to a limited degree apoptosis of PV CD34⁺ cells, whereas Nutlin-3 combined with Peg IFN-α 2a induced apoptosis in PV CD34⁺ cells to a far greater extent. *P < .05. **P < .01. n = 4.

Figure 5

Low doses of Peg IFN-α 2a and Nutlin-3 increase p53 activity. Western blotting demonstrated that the phospho-p53 level in PV CD34⁺ cells from 2 individual patients (PV1 and PV2) was increased after treatment with low doses of Nutlin-3 combined with Peg IFN-α 2a. The p21 protein level was also increased by combination drug therapy.

Figure 6

Low doses of Peg IFN-α 2a and Nutlin-3 activate p53 pathway through different mechanisms. (A) Peg IFN-α 2a, but not Nutlin-3, increased p-STAT1 in CD34⁺ cells. Treatment with Nutlin-3 or Peg IFN-α 2a alone for 48 hours led to increased cytoplasmic p21, PUMA, and Bak protein levels; treatment with the 2 drugs in combination led to an even greater increase in p21, PUMA and Bak levels. (B) The quantification of cytoplasmic levels of p21, PUMA, and Bak was determined densitometrically and normalized to GAPDH.